Recent developments have shown the potential of Optical Imaging (OI) for medical imaging of tumors and other disease manifestations. A substantial benefit of OI is the simplicity of the instrumentation required. The equipment is small, portable, and relatively inexpensive. A typical OI system consists of a filtered excitation light source (laser or otherwise), a CCD camera to record the image, and a computer to analyze and display the data. To enhance the quality of imaging, fluorescent bio-conjugates have been employed as "contrast media". In a number of recent research reports, these agents have been shown to have remarkable value for selective image enhancement of tissues, and potentially for viewing biochemical events in the cell (Molecular Imaging). In particular, fluorescent reporters in the near infrared (NIRF or near infra red fluorescence, emission approximately 700nm-1000nm) have shown excellent signals and low background even from tissues several centimeters deep. It has become clear that the potential of OI is fundamentally dependent on the photophysical performance of NIRF probes as contrast media. A powerful strategy for OI probe design is to adapt recently gained knowledge from the engineering of quenched probes for genetic assays. Here, fluorescent probes are designed to be turned off by a suitably placed "dark quencher" (non-fluorescent quencher), and fluoresce as a result of a probe cleavage or receptor binding that spatially separates the reporter and quencher. Successful design of dark quenched fluorescent probes requires careful selective pairing of fluorophore and quencher to minimize background noise. To date, quenching of NIR dyes has been via dye-dye interactions between fluorescent pairs, which are inherently inferior to dark quenched strategies. In Phase I of this grant we will systematically evaluate quenching of NIR dyes with a battery of different dark quenchers, including our proprietary Black Hole Quenchers, to determine optimal dye-quencher parings for OI contrast media design. Both FRET and "static" quenching modes will be evaluated. In Phase II, we will apply our knowledge to design a variety of contrast media NIRF probe candidates. Design strategies will be based on peptide, antibody, and oligonucleotide carders. Finally, candidate probes will be evaluated, in vivo, under OI conditions in a disease mouse animal model.